Nicotine addiction remains a major health problem in the US and throughout the world. The rewarding effects of nicotine are well-documented, but higher doses of nicotine have intensely aversive effects. Initial responses to nicotine can predict future dependence (i.e., subjects whose first nicotine experiences were rewarding are more likely to become nicotine dependent relative to subjects whose first nicotine experiences were aversive). Thus, the balance between nicotine reward and aversion likely contributes to the development and maintenance of nicotine addiction. Despite considerable understanding of the effects of nicotine on reward-related circuitry, treatments for nicotine dependent populations remain limited. Understanding the mechanisms that underlie aversion to nicotine can provide novel insights into the factors that contribute to nicotine dependence. These insights have the potential to reveal novel therapeutic targets and strategies. Recently, the projection from the medial habenula (MHb) to the interpeduncular nucleus (IPN) was shown to mediate nicotine aversion. Suppressing MHb-IPN activity reduces the aversive effects of nicotine, while enhancing MHb-IPN activity enhances aversion to nicotine. Although these results implicate the MHb-IPN circuitry, the downstream post-synaptic targets of the IPN and the neurotransmitters involved remain largely uncharacterized. Some evidence suggests that this circuitry ultimately suppresses VTA dopamine (DA) neurons. Burst activity in DA neurons has been linked to motivated behaviors, and suppression of DA neuron output results in an aversive experience. While the IPN projects to several brain areas, it strongly innervates the lateral dorsal tegmental nucleus (LDTg), a brainstem cholinergic center that controls burst firing of VTA DA neurons. Activation of LDTg neurons that project to the VTA results in enhanced activity in the VTA as well as conditioned place preference, whereas inhibition of VTA DA neurons has been linked to conditioned place aversion. Therefore, inhibiting the LDTg (via IPN excitation), and consequently VTA DA neurons, may diminish the rewarding effects of nicotine. Experiments outlined in this proposal will examine the cellular mechanisms underlying the aversive effects of nicotine and how this aversion impacts reward circuitry. We will use optogenetic strategies to explore the cellular mechanisms mediating the communication between the MHb-IPN pathway and VTA DA neurons. Causal links between these pathways and nicotine-induced behaviors will be explored by optogenetic excitation or inhibition of these pathways while testing nicotine-related behaviors. Exploring the mechanisms by which the MHb-IPN circuitry mediates nicotine-aversion could yield novel therapeutic targets and treatment strategies for helping smokers quit successfully.